Review Comparative Pathobiology of the Intestinal Protozoan Parasites lamblia, Entamoeba histolytica, and parvum

Andrew Hemphill *, Norbert Müller and Joachim Müller

Institute of , University of Berne, Länggass-Strasse 122, 3012 Berne, Switzerland * Correspondence: [email protected]; Tel.: +41-31-631-23-84

 Received: 1 July 2019; Accepted: 25 July 2019; Published: 29 July 2019 

Abstract: Protozoan parasites can infect the human intestinal tract causing serious diseases. In the following article, we focused on the three most prominent intestinal protozoan pathogens, namely, Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum. Both C. parvum and G. lamblia colonize the duodenum, jejunum, and ileum and are the most common causative agents of persistent diarrhea (i.e., cryptosporidiosis and ). Entamoeba histolytica colonizes the colon and, unlike the two former pathogens, may invade the colon wall and disseminate to other organs, mainly the liver, thereby causing life-threatening amebiasis. Here, we present condensed information concerning the pathobiology of these three diseases.

Keywords: diagnosis; immunopathology; intestinal infections; pathogens; treatment

1. Introduction Besides bacterial and viral pathogens, protozoan parasites can also infect the human intestinal tract and cause serious diseases [1]. In this article, we will focus on the three most relevant protozoal pathogens, namely, Cryptosporidium parvum (and closely related ), Giardia lamblia, and Entamoeba histolytica. We regard these organisms as obligate pathogens because they may cause symptoms in otherwise completely healthy individuals and disappear after clearance by the and/or successful chemotherapy. Conversely, opportunistic pathogens are found in healthy individuals as a part of the normal microbiome and cause symptoms in challenged individuals only [2]. A (controversial) example is Blastocystis hominis, one of the most frequent isolated from feces [3] and pathogenic in immunocompromised and irritable bowel syndrome (IBS) patients [4]. Microsporidia belong to the kingdom of fungi [5] and would be the topic of a more detailed review on systemic mycoses. Giardia lamblia and C. parvum are the most common pathogenic intestinal protozoan parasites and causative agents of persistent diarrhea in humans [6]. In the United States and in Europe, the annual incidences for the two parasitoses are around 104 each. Entamoeba histolytica causes life-threatening amebiasis after invasion of the colon wall and advancement to the liver and other organs. In the EU and the US, most cases of amebiasis are associated with travelers coming from endemic areas. Worldwide, the annual incidence is estimated at around 100 million individuals (Table1). The three intestinal protozoans discussed in this review have very simple biological cycles. There are no intermediate hosts. Cysts or oocysts (Cryptosporidium) are excreted in feces and (auto) infection occurs via ingestion of these permanent stages (Figure1). As a consequence, due to the poor hygienic conditions, the prevalence of these three intestinal protozoans is, however, much higher in underdeveloped countries thereby constituting a major problem for global health (see Table2 for an overview).

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Table 1. causing intestinal infections. The protozoa presented in this review are in bold. Pathogens 2019, 8, 116 2 of 21 Incidence Classification Species Pathogenicity Localization (Super Groups) World EU US 2 Table 1. Protozoa causing intestinal1 infections.3 The protozoa presented in this review are in bold. Cliliata Balantidium coli Rare colon Classification(Diaphoretickes) Incidence Species Pathogenicity Localization (Super Groups) World 1 US 2 EU 3 Stramenopile Blastocystis sp. Cliliata Very high opportunistic (?) colon Balantidium coli (Diaphoretickes) Rare colon (Diaphoretickes) Cryptosporidium duodenum, jejunum, Stramenopile nk 8–9 7 obligate Blastocystis sp. parvum (Diaphoretickes) Very high opportunistic (?)ileum colon (Diaphoretickes) Cryptosporidium ApicomplexaTrichomonadina unclear, most likely same as G. duodenum, jejunum, nkCommon 8–9 7 obligate colon parvum (Diaphoretickes)() lamblia ileum

Dientamoeba TrichomonadinaAmoebozoa unclear, most likely same as Entamoeba histolytica 100 rareCommon rare obligate colon, liver colon fragilis (Excavata)(Amorphea) G. lamblia Entamoeba Amoebozoa Diplomonadida 100 rare rare obligateduodenum, jejunum, colon, liver histolyticaGiardia lamblia(Amorphea) 250 15 18 obligate (Excavata) ileum Diplomonadida duodenum, jejunum, Giardia lamblia 250 15 18 obligate (Excavata)Fungi ileum Microsporidia sp. Very high opportunistic colon (Amorphea) Fungi Microsporidia sp. Very high opportunistic colon 1 WHO (World(Amorphea) health organization), NIH (National Institute of Health) (×106/year); 2 CDC (Center of 1 WHO (WorldDisease health Control), organization), data for 2011–2012; NIH (National 3 ECDC (European Institute ofCenter Health) of Disease ( 106 /Control),year); 2 CDCdata for (Center 2014– of Disease Control), data2015 for (both 2011–2012; ×103/year).3 ECDC nk, not (European known. We Centerbsites: of ecdc.europ Disease Control),a.eu; www.cdc.gov; data× for 2014–2015 www.nlm.nih.gov; (both 10 3/year). nk, × not known.www.who.int. Websites: ecdc.europa.eu ; www.cdc.gov; www.nlm.nih.gov; www.who.int.

Figure 1. Simplified biological cycle of Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum. Figure 1. Simplified biological cycle of Giardia lamblia, Entamoeba histolytica, and Cryptosporidium Table 2. Overviewparvum. of diseases caused by the protozoans presented in this review; see Reference [1] and text of this review for further references. As a consequence, due to the poor hygienic conditions, the prevalence of these three intestinal protozoans is, however,Giardiasis much higher in underde Amebiasisveloped countries thereby constituting Cryptosporidiosis a major problemGiardia for lambliaglobal health (see Table 2 Entamoeba for an overview). histolytica Cryptosporidium parvum Transmission Via (Oo)cysts in feces Symptoms Persistent diarrhea (>1 w), Mild-to-acute diarrhea, nausea, abdominal pain, Acute Diarrhea, abdominal pain. malabsorption. low-grade fever. Malabsorption, loose stools, Severe diarrhea, vomiting, malabsorption, volume Fever, sepsis, liver abscesses, skin Chronic gassiness, cramping, fatigue, liver or depletion and wasting, biliary and respiratory lesions. pancreatic inflammations. involvement in immunodeficient persons. Diagnosis Feces Microscopy (cysts), coproantigen Microscopy (trophozoites, cysts), Microscopy, coproantigen test, PCR, Biopsy material test, PCR. coproantigen test and PCR. enzyme-immunoassays. Positive in the case of Serology extraintestinal infection. Giardiasis, Rotavirus, Cyclospora cayetanensis, Clostridium Differential Cryptosporidiosis, IBS, celiac. IBD, cancer, bacterial infections. difficile. Diagnosis Microsporidia, IBS, celiac. Management Immunocompetent: NTZ ()100–500 mg p.o. twice daily, 3 d. First line treatment Metronidazole (500 to 750 mg p.o. t.i.d., 10 d) HIV: Antiretroviral therapy, possibly combined with NTZ or paromomycin/azithromycin. Other immunodeficiencies: NTZ 500 mg twice daily, 14 d. Prevention Personal hygiene, water treatment, appropriate cleaning and storage of vegetables. Pathogens 2019, 8, 116 3 of 21

2. Etiology and Epidemiology

2.1. Giardia lamblia Giardia lamblia is an anaerobic, but to some extent also aerotolerant, with several prokaryotic properties [7–9] belonging to the phylum Diplomonadida, super-group Excavata [5]. Giardia exists in two morphologic forms: the multi-flagellated trophozoite (four pairs of flagella) and the cyst. The trophozoite is dinucleated, pear-shaped, multi-flagellated, 9 to 15 µm long, 5 to 15 µm wide,Pathogens and 2 to2019 4, µ8, m116 thick, with an adhesive disk on the ventral surface (Figure2). 4 of 21

FigureFigure 2. Giardia 2. Giardia lamblia lambliatrophozoites trophozoites and and cysts cysts visualizedvisualized by by SEM. SEM. (A ()A trophozoites) trophozoites cultured cultured in axenic in axenic in vitroin vitroculture, culture, exposing exposing either either their their dorsal dorsal surface surface (d)(d) or ventral ventral disc disc (vd). (vd). Note Note the themultiple multiple flagella flagella (f). Bar(f). Bar= 8 =µ 8m. µm. (B ()B) Trophozoite Trophozoite (T) and and cyst cyst (C) (C) stages stages in a in mouse a mouse feces fecessample. sample. Bar = 6.4 Bar µm.= (6.4C) µm. (C) TrophozoitesTrophozoites attachingattaching to to the the inte intestinalstinal surface surface of an of experimenta an experimentallylly infected infected mouse. Bar mouse. = 8 µm. Bar (D=,E)8 µm. (D,E)Higher Higher magnification magnification view view of ofa trophozoite a trophozoite attach attachinging to the to themouse mouse intestinal intestinal surface surface and to and human to human coloncolon carcinoma carcinoma cells cells (Caco2) (Caco2) during duringin in vitro vitroculture, culture, respectively. Arrows Arrows de delineatelineate the theperiphery periphery of of the ventralthe ventral disc. disc. Bar Bar in (inD ,(ED),E=) 4= 4µ m.µm.

Trophozoites2.2. Entamoeba livehistolytica attached on dudodenal and jejunal epithelial cells and thrive on nutrients from the intestinalHuman fluid amebiasis with amino is caused acids, by especially E. histolytica arginine (Amoebozoa, as their preferredAmorphae fuela). All [10 ,pathogenic11]. Detached Entamoeba are classified as E. histolytica, whereas the species Entamoeba dispar comprises non- pathogenic Entamoeba strains [22]. As for G. lamblia, two stages can be distinguished, namely, trophozoites and cysts. The motile, mononucleated trophozoite (10 to 20 µm ᴓ, sometimes larger) colonizes the colon (and eventually other organs), where it may transform into the cyst stage having a similar size, but one to four nuclei (Figure 3).

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trophozoites form quadrinucleated, thick-walled cysts (8–10 µm in diameter). The cysts are excreted in the feces and constitute the infectious stage. Thus, giardiasis is caused by fecal contaminations of [12], food [13], or direct contact with feces [14], waterborne transmission being regarded as a major source [15]. Pathogenesis and virulence depend on the genotype of the Giardia Figure 2. Giardia lambliastrain trophozoites and the and immune cysts visualized and nutritional by SEM. status(A) trophozoites of the . cultured To date, in axenic eight genetic assemblages (A to H) in vitro culture, exposinghave either been their described. dorsal surface It is (d) or ventral established disc (vd). that Note isolates the multi fromple assemblages flagella A and B cause infection (f). Bar = 8 µm. (B) Trophozoitein humans (T) [ 16and–18 cyst]. There(C) stage is,s however,in a mouse morefeces sample. recent evidenceBar = 6.4 µm. that (C isolates) from assemblage E are Trophozoites attachingpathogenic to the intestinal for humans,surface of an as experimentally well [19]. Since infected these strainsmouse. Bar can = be 8 µm. found (D,E in) humans as well as in animals, Higher magnification viewgiardiasis of a tro canphozoite be regarded attaching asto athe mouse intestinal [20]. Especially, surface and young to human children living in poor sanitary colon carcinoma cells (Caco2)conditions during are in exposed vitro culture, to giardiasis respectively which—in. Arrowscombination delineate the periphery with malnutrition of or immunosuppression the ventral disc. Bar in (e.g.,(D,E) HIV)—can= 4 µm. be fatal. Moreover, persistent infections in children may cause stunting [21].

2.2. Entamoeba histolytica 2.2. Entamoeba histolytica Human amebiasis is causHumaned by amebiasis E. histolytica is caused (Amoebozoa, by E. histolytica Amorphaea(Amoebozoa,). All Amorphaea). pathogenic All pathogenic Entamoeba Entamoeba are classified are as classifiedE. histolytica as E., histolytica whereas , thewhereas species the speciesEntamoebaEntamoeba dispar disparcomprisescomprises non- non-pathogenic Entamoeba pathogenic Entamoeba strainsstrains [22] [22. ]. As As for for G.G. lamblia lamblia, , two two stages stages can can be be distinguished, distinguished, namely namely,, trophozoites and cysts. trophozoites and cysts. TheThe motile, motile, mononucleated mononucleated trophozoite trophozoite (10 (10 to to 20 20 µmµm ᴓ, sometimes larger) colonizes the colon (and colonizes the colon (and eventuallyeventually other other organs), organs), where where it it may may transform transform into into the the cyst cyst stage stage having having a similar size, but one to a similar size, but one to fourfourPathogens nuclei nuclei 2019 (Figure (Figure, 8, 116 33).). 5 of 21

Figure 3. Scanning electron microscopy of in vitro-cultured Entamoeba histolytica trophozoites. Note the Figure 3. Scanning electron microscopy of in vitro-cultured Entamoeba histolytica trophozoites. Note different shapes and cell surface structures such as rough (r) and smooth (s) adopted by the trophozoites, the different shapes and cell surface structures such as rough (r) and smooth (s) adopted by the and the cytoplasmic protrusions mediating contact to the surface (arrows). Bar in (A,B) = 5 µm. trophozoites, and the cytoplasmic protrusions mediating contact to the surface (arrows). Bar in (A,B) Human= 5 µm. infection occurs via the ingestion of excreted cysts, thus via fecal–oral contamination upon waterborne, foodborne, or person-to-person transmission. Therefore, poor sanitation and overcrowdingHuman infection are socio-economic occurs via factorsthe ingestion favoring of amebiasis. excreted cysts, In the EUthus and via the fecal–oral US, nowadays, contamination cases of amebiasisupon waterborne, are mostly foodborne, associated or to person-to-person travelers coming fromtransmission. endemic Therefore, areas. Amebiasis poor sanitation is still a major and causeovercrowding of morbidity are socio-economic and mortality in factors developing favoring countries amebiasis. [23]. In the EU and the US, nowadays, cases of amebiasis are mostly associated to travelers coming from endemic areas. Amebiasis is still a major 2.3.cause Cryptosporidium of morbidity and sp. mortality in developing countries [23]. The genus Cryptosporidium is classified into the phylum Apicomplexa, class , and order 2.3. Cryptosporidium sp. . More recent studies, however, indicate, that this genus is more closely related to GregarinesThe genus [24]. Cryptosporidium Currently, 31 valid is classifiedCryptosporidium into thespecies phylum have Apicomplexa, been recognized class in Conoidasida, fish, amphibians, and reptiles,order Eucoccidiorida. birds, and mammals, More recent and studies, an additional however, 40 distinctindicate, genotypes that this genus from ais varietymore closely of vertebrate related hoststo Gregarines are described. [24]. Currently, Nearly 20 31 species valid and Cryptosporidium genotypes have species been have reported been in recognized humans, of in which fish, C.amphibians, hominis and reptiles,C. parvum birds,account and mammals, for >90% and of allan additional cases [25]. 40Cryptosporidium distinct genotypes hominis fromtransmission a variety of occursvertebrate via humans,hosts are whiledescribed.C. parvum Nearlyhas 20 a species high zoonotic and genotypes potential. have There been is stillreported a lack in of humans, subtyping of which C. hominis and C. parvum account for >90% of all cases [25]. Cryptosporidium hominis transmission occurs via humans, while C. parvum has a high zoonotic potential. There is still a lack of subtyping tools for many Cryptosporidium species of public and veterinary health importance, and the genetic determinants of host specificity of Cryptosporidium species are only poorly understood. Diarrhea is caused through mechanisms involving increased intestinal permeability, chloride , and malabsorption. Although otherwise healthy individuals can acquire infection, other conditions such as immunodeficiency caused by HIV infection, malnutrition, chemotherapy, diabetes mellitus or bone marrow or solid organ transplantation constitute increased risks for more severe and disseminated disease [26]. Livestock, particularly , are important reservoirs of zoonotic infections [27]. The impact is especially devastating in infants in the resource-constrained regions, and disease is associated with an estimated annual death rate of >200,000 children below 2 years of age. An epidemiological study of over 22,000 infants and children in Africa and Asia [28] found that Cryptosporidium was one of the four pathogens responsible for most of the severe diarrhea and was considered the second greatest cause of diarrhea and death in children after Rotavirus [29]. Common means of transmission of cryptosporidiosis is by municipal drinking water and water in swimming pools and via contaminated food. A high risk of infection concerns child care workers, parents of infected children, people who handle infected animals, those exposed to human feces through sexual contact, and healthcare providers as well as pregnant women and individuals suffering from immunodeficiency [26]. Cryptosporidium has a monoxenous life cycle [30]. Infection takes place via oral ingestion of oocysts containing invasive sporozoites. These sporozoites enter intestinal epithelial cells and form a

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tools for many Cryptosporidium species of public and veterinary health importance, and the genetic determinants of host specificity of Cryptosporidium species are only poorly understood. Diarrhea is caused through mechanisms involving increased intestinal permeability, chloride secretion, and malabsorption. Although otherwise healthy individuals can acquire infection, other conditions such as immunodeficiency caused by HIV infection, malnutrition, chemotherapy, diabetes mellitus or bone marrow or solid organ transplantation constitute increased risks for more severe and disseminated disease [26]. Livestock, particularly cattle, are important reservoirs of zoonotic infections [27]. The impact is especially devastating in infants in the resource-constrained regions, and disease is associated with an estimated annual death rate of >200,000 children below 2 years of age. An epidemiological study of over 22,000 infants and children in Africa and Asia [28] found that Cryptosporidium was one of the four pathogens responsible for most of the severe diarrhea and was considered the second greatest cause of diarrhea and death in children after Rotavirus [29]. Common means of transmission of cryptosporidiosis is by municipal drinking water and water in swimming pools and via contaminated food. A high risk of infection concerns child care workers, parents of infected children, people who handle infected animals, those exposed to human feces through sexual contact, and healthcare providers as well as pregnant women and individuals suffering from immunodeficiency [26]. Cryptosporidium has a monoxenous life cycle [30]. Infection takes place via oral ingestion of Pathogens 2019, 8, 116 6 of 21 oocysts containing invasive sporozoites. These sporozoites enter intestinal epithelial cells and form parasitophorousa parasitophorous vacuole vacuole (PV) (PV) that that is islocated located at at the the apical apical part part of of the the host host cell, cell, just just underneath underneath the brushbrush border (Figures 44 andand5 5).).

Figure 4. Light microscopy of Cryptosporidium.(A–C) Histological sections of paraffin-embedded C. Figureparvum- 4.infected Light microscopy intestinal tissue, of Cryptosporidium stained with. hematoxylin (A–C) Histological and eosin. sections (A) A of low-magnification paraffin-embedded view, C. parvum-where theinfected boxed intestinal area in( tissue,A) is magnified stained with in (hematoxylinB). Arrows point and eosin. towards (A) C.A low-magnification parvum parasitophorous view, wherevacuoles the seen boxed as area round in bodies(A) is magnified on the surface in (B). of Arrows the epithelial point towards layer. (D C.) Modifiedparvum parasitophorous Ziehl–Neelsen vacuolesstaining ofseen oocysts as round (red, bodies circular on bodies) the surface in a stool of the sample. epithelial Bar inlayer. A = (1450D) Modifiedµm; B and Ziehl–Neelsen C = 260 µm; stainingD = 7.5 µ ofm. oocysts (red, circular bodies) in a stool sample. Bar in A = 1450 µm; B and C = 260 µm; D = 7.5 µm.

Pathogens 2019, 8, 116 6 of 21 parasitophorous vacuole (PV) that is located at the apical part of the host cell, just underneath the brush border (Figures 4 and 5).

Figure 4. Light microscopy of Cryptosporidium. (A–C) Histological sections of paraffin-embedded C. parvum-infected intestinal tissue, stained with hematoxylin and eosin. (A) A low-magnification view, where the boxed area in (A) is magnified in (B). Arrows point towards C. parvum parasitophorous vacuoles seen as round bodies on the surface of the epithelial layer. (D) Modified Ziehl–Neelsen staining of oocysts (red, circular bodies) in a stool sample. Bar in A = 1450 µm; B and C = 260 µm; D = Pathogens7.5 µm.2019 , 8, 116 6 of 21

Figure 5. Transmission electron microscopy (TEM) of Cryptosporidium parvum. Madin Darbey canine

kidney (MDCK) cells were infected with C. parvum sporozoites and fixed and processed for TEM after 72 h of culture. Sporozoites have formed parasitophorous vacuoles (PVs) on the apical part of the MDCK cells, occupying a space which is still intracellular, but essentially extra-cytoplasmatic, giving rise to meronts. (B) A low magnification view of three PVs, with two developing meronts (me) clearly visible. Arrows indicate the outer host cell surface membrane. Bar = 12 µm. (C) A higher magnification view of the boxed area in (A). Asterisks (*) indicate the membrane of the parasitophorous vacuole, arrows point towards the host cell surface membrane, mn indicates nuclei of developing merozoites. Note the electron-dense zone where the PV is in close contact to the host cell cytoplasm, formed due to the cytoskeletal rearrangements. Bar = 2.5 µm.

These sporozoites will then develop into merozoites, which either undergo asexual proliferation, egress, and re-infection of other intestinal cells (named type I merozoites), or develop into type II merozoites and undergo sexual development, leading to the formation of a zygote, which produces an oocyst wall and infective sporozoites. In the case that the oocysts are thin-walled, excystation of sporozoites can take place already in the intestine, leading to auto-infection, while thick-walled oocysts are excreted at very large numbers and are infectious upon oral ingestion. Oocysts are environmentally stable and can survive for many months under temperate and moist conditions, and they are resistant to chlorine levels usually applied in water treatment.

3. Pathogenicity and Virulence

3.1. General Remarks The virulence of an intestinal pathogen results from its own genetic background, the competence of the host immune system, its nutritional status (e.g., the acquisition of iron [31]) and the interaction with other intestinal (Figure6). Pathogens 2019, 8, 116 7 of 21

Figure 5. Transmission electron microscopy (TEM) of Cryptosporidium parvum. Madin Darbey canine kidney (MDCK) cells were infected with C. parvum sporozoites and fixed and processed for TEM after 72 h of culture. Sporozoites have formed parasitophorous vacuoles (PVs) on the apical part of the MDCK cells, occupying a space which is still intracellular, but essentially extra-cytoplasmatic, giving rise to meronts. (B) A low magnification view of three PVs, with two developing meronts (me) clearly visible. Arrows indicate the outer host cell surface membrane. Bar = 12 µm. (C) A higher magnification view of the boxed area in (A). Asterisks (*) indicate the membrane of the parasitophorous vacuole, arrows point towards the host cell surface membrane, mn indicates nuclei of developing merozoites. Note the electron-dense zone where the PV is in close contact to the host cell cytoplasm, formed due to the cytoskeletal rearrangements. Bar = 2.5 µm.

These sporozoites will then develop into merozoites, which either undergo asexual proliferation, egress, and re-infection of other intestinal cells (named type I merozoites), or develop into type II merozoites and undergo sexual development, leading to the formation of a zygote, which produces an oocyst wall and infective sporozoites. In the case that the oocysts are thin-walled, excystation of sporozoites can take place already in the intestine, leading to auto-infection, while thick-walled oocysts are excreted at very large numbers and are infectious upon oral ingestion. Oocysts are environmentally stable and can survive for many months under temperate and moist conditions, and they are resistant to chlorine levels usually applied in water treatment.

3. Pathogenicity and Virulence

3.1. General Remarks The virulence of an intestinal pathogen results from its own genetic background, the competence Pathogensof the host2019 immune, 8, 116 system, its nutritional status (e.g., the acquisition of iron [31]) and the interaction7 of 21 with other intestinal microorganisms (Figure 6).

Figure 6. PathogenicityPathogenicity and and virulence. Pathogenicity Pathogenicity an andd virulence virulence are are the the result of interactions between the genotype of the pathogen, the immune re response,sponse, the nutritional status, and the intestinal microbiome of the host.

All three parasites discussed here in detail infect their hosts via ingested cysts and colonize the digestive tract. They attach to the epithelial surface of the duodenum/ileum (Cryptosporidium sp.,

Giardia lamblia) or colon (Entamoeba histolytica) and elicit an immune response involving interleukin (IL)-6 production by T-cells, dendritic cells and mast cells. Interleukin-6 stimulates IL-17-mediated host defense (production of intestinal IgA (Immunoglobulin A) and anti-microbial peptides). Furthermore, mast cell degranulation promotes peristalsis. The resulting inflammatory reactions (see Reference [32] for review) are more (E. histolytica) or less (G. lamblia) pronounced. Thus, immunopathology may play an important role in the case of E. histolytica, where inflammatory reactions may facilitate the penetration of the colon wall and the subsequent systemic spreading causing amebiasis. In the case of giardiasis and cryptosporidiosis, inflammatory reactions are much less pronounced.

3.2. Giardia lamblia Unlike other protozoal pathogens, G. lamblia trophozoites and their conversion into cysts can be easily studied in vitro, the genome is sequenced (GiardiaDB.org), molecular genetics are well established, and in vivo-models (e.g., orally cyst-infected mice) are available. Nevertheless, to date, the pathophysiology of giardiasis is not entirely understood [33]. When ingested cysts reach the stomach, their cyst wall is digested and they transform into trophozoites colonizing the of the duodenum and the proximal jejunum. As a first reaction on part of the host, cells in contact with the trophozoites may undergo apoptosis, epithelial tight junctions are ruptured [34], and CD8+-lymphocytes are activated [35]. As a consequence, brush-border microvilli are shortened [35], resulting in deficiencies in disaccharidases and other enzymes [36]. In a next step, adaptive immune responses are elicited via IL-6-producing dendritic and mast cells [37] and CD4+ T-cells producing IL-17 and TNFalpha [38]. Another potential source of IL-17 are tuft cells that may be stimulated by metabolites excreted by trophozoites thereby enhancing the pro-inflammatory reactions in the intestinal epithelium, as found for closely related and for helminths [39]. As a consequence, Pathogens 2019, 8, 116 8 of 21 cytotoxic [40] secretory IgA and defensins are produced resulting in the elimination of trophozoites from the intestinal surface. Moreover, nitric oxide (NO) produced by epithelial cells and immune cells inhibits trophozoite proliferation [33,41] and—NO production in neurons—in combination with mast cell degranulation—promotes intestinal peristalsis thus contributing to the expulsion of trophozoites. Although minor, the intestinal microbiota may increase the efficiency of the anti-giardial immune response. In fact, trophozoites may induce dysbiosis of the microbiota resulting in an immunological effect supporting infection [42] (Figure7). Recent studies consider cysteine proteases involved in pathogenesis, disruption of intestinal epithelial junctions, apoptosis of intestinal epithelial cells, and also the degradation of host immune factors (e.g., immunoglobulins and chemokines), mucus depletion, and microbic dysbiosis as major virulence factors [18]. Since IgA recognizes surface as predominant antigens, G. lamblia has developed an escape strategy based on the variation of these surface proteins. According to a generally admitted hypothesis, only one (major) variant surface (VSP) is expressed on a single trophozoite [43]. The expression of different VSPs—and thus antigenic variation—is triggered by epigenetic mechanisms involving changes in the chromatin state [44] and/or RNA interference [45,46]. Trophozoites surviving the exposure to IgA react by expressing different variants of these so-called “variant surface proteins” thereby escaping the immune response [47]. This strategy is called “antigenic switch” (Figure8). Antigenic switch does not occur only as a reaction to exposure to antibodies, but also to drugs [48–50] and is—in all likelihood—epigenetically regulated [51]. Recent results suggest that VSPs play not only a passive role in escaping host immune responses but also may actively participate in damaging epithelial cells via proteolytic activities [52]. An excellent review on parasitic strategies to Pathogenscircumvent 2019, host8, 116 immune reactions is given elsewhere [32]. 9 of 21

FigureFigure 7. 7. IntestinalIntestinal colonization colonization of of GiardiaGiardia lamblia lamblia (Gl)(Gl) and and host host defense defense mechanisms mechanisms against against the the parasite.parasite. Explanation Explanation see see text. text.

Figure 8. Illustration of the fundamental paradigm of antigenic variation in Giardia lamblia. (A) Trophozoites are stained with a monoclonal antibody directed against the variant surface protein

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Figure 7. Intestinal colonization of Giardia lamblia (Gl) and host defense mechanisms against the Pathogensparasite.2019, 8 Explanation, 116 see text. 9 of 21

Figure 8. Illustration of the fundamental paradigm of antigenic variation in Giardia lamblia. Figure 8. Illustration of the fundamental paradigm of antigenic variation in Giardia lamblia. (A) (A) Trophozoites are stained with a monoclonal antibody directed against the variant surface protein Trophozoites are stained with a monoclonal antibody directed against the variant surface protein (VSP) H7 (green staining). The presence of trophozoites is visualized by the characteristic staining of the double nuclei. After in vivo culture and re-isolation, the green staining is lost, meaning that new VSPs have replaced VSP H7. (B) Is one VSP replaced by another VSP or by several different VSPs thereby increasing the heterogeneity of the trophozoite population? Transcriptional studies have revealed that after subsequent in vivo cultivation of G. lamblia clone H7, the variability of VSPs increases.

3.3. Entamoeba histolytica Entamoeba histolytica trophozoites are easily cultivated in vitro, the genome has been sequenced (AmoebaDB.org), and susceptible and resistant rodent in vivo models are available, thus allowing experimental investigations of virulence factors [53,54]. While passing the digestive tract, ingested cysts liberate trophozoites proliferating within the colon. Unlike Giardia, they dwell not only on intestinal fluids but produce cysteine proteases and Gal/GalNAc-lectins both damaging the intestinal mucosa by structural destabilization and cellular destruction of the epithelial cell layer. As a consequence, E. histolytica penetrates the intestinal mucosa by evading and, at the same time, exploiting the mucosal immune response of the host. In an initial phase, mucosal inflammation is promoted by secretion of E. histolytica macrophage migration inhibitory factor (EhMIF). Supported by tissue-destructive and cytolytic effectors such as matrix metalloproteinases (MMPs) and oxygen free radicals (ROS) produced by infiltrating inflammatory cells, focal perforation of the intestinal mucosa may occur allowing the trophozoites to invade the colon wall [55,56]. Infecting E. histolytica trophozoites, however, have to face different host defense mechanisms, namely, (i) increased mucus production protecting the epithelial surface; (ii) secretion of defensin 2 and pro-inflammatory cytokines (Il-1β, IL-8, TNF-α) after contact of trophozoites with epithelial cells [57]; (iii) Th1-mediated immune responses during acute amebiasis, and Th2- and Th17-mediated Pathogens 2019, 8, 116 10 of 21

immune responses during chronic amebiasis [58]. Consequently, neutrophils are attracted via IL-8 and macrophages via IL-1β. IL-17 production favors persistence of infection as shown by comparing IL-17-knock-out to wild-type mice [59]. Since E. histolytica trophozoites resist killing by neutrophils, the resulting inflammatory reaction even enhances tissue injury thereby promoting the infection [60]. In 90% of patients, the colon wall is not invaded and the infection remains asymptomatic or with mild symptoms. In the remaining 10%, the colon barrier is broken, and trophozoites spread into the wall and surrounding tissues causing local necrosis and ulcer formation. Once the colon wall is invaded, however, amebiasis may spread hematogenously to any organ in the body, most commonly the liver and the lungs [60,61]. It is still unclear to which extent host cells are directly involved in the destruction of the colon wall. There is some evidence from in vivo models that the inflammatory reactions of host cells and not proteolytic degradation of the wall by the parasite is responsible for tissue damage, but it is difficult to extrapolate from defined animal models to the situation in patients with diverse physiological backgrounds and immune status [62]. Another intriguing aspect is the interaction with the colon microbiome. Enteric bacteria may stimulate the oxidative stress defenses of E. histolytica [63]. Moreover, there are good indications that E. histolytica causes dysbiosis of the colon microbiome stimulating immune responses facilitating systemic invasion [64]. It is still unclear to which extent this parasite evades or suppresses [65] host

Pathogensimmune 2019 responses, 8, 116 during this systemic spread (Figure9). 11 of 21

FigureFigure 9. Intestinal colonizationcolonization and and invasion invasion of E.of histolyticaE. histolytica(Eh) (Eh) and hostand defensehost defense mechanisms mechanisms against againstthe parasite. the parasite. For explanations For explanations see text. see text. 3.4. Cryptosporidium sp. 3.4. Cryptosporidium sp. Unlike G. lamblia and E. histolytica, Cryptosporidium sp. (and other closely related Coccidia, e.g., Unlike G. lamblia and E. histolytica, Cryptosporidium sp. (and other closely related Coccidia, e.g., Eimeria sp.) cannot be cultivated axenically [66]. The genome has been sequenced (CryptoDB.org), Eimeria sp.) cannot be cultivated axenically [66]. The genome has been sequenced (CryptoDB.org), but genetic manipulation of this protozoal parasite [67] has only recently been established using but genetic manipulation of this protozoal parasite [67] has only recently been established using CRiSPR/Cas-mediated gene targeting [68]. The studies leading to the identification of pathogenicity CRiSPR/Cas-mediated gene targeting [68]. The studies leading to the identification of pathogenicity and virulence factors are performed in coculture systems [69,70] with intestinal [71], biliary [72], and virulence factors are performed in coculture systems [69,70] with intestinal [71], biliary [72], tracheal [73] or esophagus [74] epithelial cell layers or organoids [75], where infection, invasion, tracheal [73] or esophagus [74] epithelial cell layers or organoids [75], where infection, invasion, and and differentiation can be studied from a couple of days until several weeks [76]. Based on studies differentiation can be studied from a couple of days until several weeks [76]. Based on studies with with these systems, factors mediating excystation, invasion, PV formation, intracellular maintenance, these systems, factors mediating excystation, invasion, PV formation, intracellular maintenance, and host cell damage could be identified as important virulence factors [77]. After oral uptake of oocysts, Cryptosporidium sporozoites hatch in the , attach to and invade host epithelial cells [78], preferentially cells in mitosis [79]. This is in good agreement with more recent findings that in pig intestinal cells infected with C. parvum, expression of genes involved in mitosis are upregulated, but neither stress- nor apoptosis-related genes [80]. Host-cell apoptosis is dimmed in early stages of infection and promoted in late stages [81–83]. In analogy to related apicomplexan parasites, it can be speculated that Cryptosporidium secretes molecules to the host cell that directly interfere with apoptosis signaling cascades at multiple levels as shown for Toxoplasma gondii [84] and for Theileria sp. [85,86]. Attachment to host cells and invasion is mediated by proteins such as a galactose/N- glactosamin-specific lectin [87] and T. gondii-SAG1 homologous proteins [88] released from a set of secretory organelles, which are constituents of the apical complex. Infection requires extensive remodeling of the [89], and invaded sporozoites are finally located in a parasitophorous vacuole, surrounded by a parasitophorous vacuole membrane (PVM) that is essentially host cell surface membrane derived and modified after invasion. Once intracellular, parasites remain at the apical domain of the host cell, therefore, the PV is an intracellular but extracytoplasmatic compartment (Figure 5). Thus, the PVM shields parasites from the host defense. Trophozoites are formed and will eventually undergo development into merozoites, as well as sexual development forming sporozoites and oocysts, which are orally infective. Tissue damage occurs through disruption of tight junctions of the epithelium, leading to cytoskeletal alterations, loss of barrier

Pathogens 2019, 8, 116 11 of 21 and host cell damage could be identified as important virulence factors [77]. After oral uptake of oocysts, Cryptosporidium sporozoites hatch in the small intestine, attach to and invade host epithelial cells [78], preferentially cells in mitosis [79]. This is in good agreement with more recent findings that in pig intestinal cells infected with C. parvum, expression of genes involved in mitosis are upregulated, but neither stress- nor apoptosis-related genes [80]. Host-cell apoptosis is dimmed in early stages of infection and promoted in late stages [81–83]. In analogy to related apicomplexan parasites, it can be speculated that Cryptosporidium secretes molecules to the host cell that directly interfere with apoptosis signaling cascades at multiple levels as shown for Toxoplasma gondii [84] and for Theileria sp. [85,86]. Attachment to host cells and invasion is mediated by proteins such as a galactose/N- glactosamin-specific lectin [87] and T. gondii-SAG1 homologous proteins [88] released from a set of secretory organelles, which are constituents of the apical complex. Infection requires extensive remodeling of the cytoskeleton [89], and invaded sporozoites are finally located in a parasitophorous vacuole, surrounded by a parasitophorous vacuole membrane (PVM) that is essentially host cell surface membrane derived and modified after invasion. Once intracellular, parasites remain at the apical domain of the host cell, therefore, the PV is an intracellular but extracytoplasmatic compartment (Figure5). Thus, the PVM shields parasites from the host defense. Trophozoites are formed and will eventually undergo development into merozoites, as well as sexual development forming sporozoites and oocysts, which are orally infective. Tissue damage occurs through disruption of tight junctions of the epithelium, leading to cytoskeletal alterations, loss of barrier function, and—ultimately—increased cell death. These alterations are caused by lytic enzymes such as phospholipases [90] and proteases [91–94]. As a response, proinflammatory cytokines like interferon gamma (IFNγ)[95], IL-6, IL-12 [96], IL-17 [97], and chemokines are released to the infection site. A detailed review is given in Reference [98] and the references therein. On the one hand, the resulting inflammatory reactions contribute to increased epithelial permeability, impaired intestinal absorption, and increased secretion thereby enhancing the symptoms like malabsorption and diarrhea. On the other hand, they stimulate innate defense mechanisms [95] such as the release of antimicrobial peptides [99] and acquired immune responses leading ultimately to the control of the infection in immunocompetent hosts (see Figure7).

4. Control and Treatment

4.1. Prevention Since all three pathogens discussed in this review are transmitted via cysts [100], the prevention is water treatment by filtration [101] or disinfection by chlorination or radiation [102]. Moreover, person-to-person and animal-to-person transmission can be prevented by standard attention to hygiene (i.e., hand washing). Treatment of asymptomatic persons excreting cysts is indicated to prevent autoinfection and the spread of infection to healthy persons.

4.2. Diagnosis The standard methodology as well as recent developments in diagnosis of intestinal parasites including G. lamblia, E. histolytica, and C. parvum have recently been summarized in the updated guidelines from the Infectious Diseases Community of Practice of the American Society of Transplantation [103]. Conventionally, diagnosis is based on the detection of trophozoites or (oo)cysts in stool samples. In addition, E. histolytica trophozoites can be identified in aspirates or biopsies sampled during colonoscopy or surgery, respectively. Coprological diagnosis of cryptosporidiosis is mostly performed by modified Ziehl–Neelsen staining of acid-fast Cryptosporidium oocysts in fecal smears (see Figure4). This method is regarded as the gold standard method for diagnosis of this protozoan parasite. While serology is considered to be of minor importance in diagnosis of giardiasis and cryptosporidiosis, ELISA tests demonstrating seroconversion as an indicator of an invasive E. histolytica infection are important tools to diagnose acute amebiasis. Pathogens 2019, 8, 116 12 of 21

Detection of ameba in stool is, however, not conclusive since non-pathogenic Entamoeba strains (E. dispar) cannot be distinguished from pathogenic strains by a mere morphological examination. This problem is solved by various E. histolytica-specific PCR tests [104,105]. A modern multiplex real-time PCR-based method allows the simultaneous detection of Giardia, Entamoeba, and Cryptosporidium in stool samples with high sensitivities and specificities [106]. These tests can be employed not only on stool, but also on biopsy material where trophozoites are rarely visible.

4.3. Treatment Like for other anaerobic pathogens, the first line treatment of amebiasis and giardiasis—as recommended by the WHO and CDC guidelines—is based on compounds containing a nitro group which is reduced, thereby forming toxic intermediates, metronidazole [107] or related nitroimidazoles being the first choice (see Table2). The nitro-thiazolide nitazoxanide is active not only against Giardia (in vitro and in vivo) but also against Entamoeba in vitro [108]. Since nitazoxanide and other thiazolides affect host cells also [109], they are not suited for the treatment of systemic infections such as amebiasis. In the case of resistance against nitro drugs which is frequent in Giardia [110], the highly effective benzimidazole may be used as a second line drug [111]. Many natural compounds, such as essential oils, some fatty acids, isoflavones, etc., inhibit the proliferation of G. lamblia trophozoites with much higher efficacy than metronidazole (see References [112] and references therein). Therefore, a diet combining low protein contents resulting in less free amino acids, especially arginine, available as fuel [11] with traditional herbs, such as basil, garlic, ginger, oregano etc., may constitute an additional management strategy, especially in the case of chronic giardiasis. Resistance to nitro drugs also occurs in E. histolytica [113]. Therefore, there is a constant interest in novel, druggable targets that are absent in the host [114]. An example for such a target is the metabolism of sulfur-containing amino acids, e.g., methionine gamma-lyase-mediated catabolism [115]. Since virulence of E. histolytica strains is directly correlated to their oxygen resistance [116], the inhibition of scavengers of reactive oxygen species such as thioredoxin reductase [117] by auranofin, as identified by an unbiased high throughput screening [118] or by related compounds, could constitute a complementary strategy [119]. Against cryptosporidiosis, chemotherapy would be valuable in immunocompromised patients, but an effective regimen has not been established [26]. Nitazoxanide and other non-nitro-thiazolides are effective against cryptosporidiosis in a rodent model [120], but it is unclear whether the compounds act on the parasite or on the host [109]. For some HIV-infected patients, paromomycin, an oral non-absorbed aminoglycoside from Streptomyces rimosus, or paromomycin combined with azithromycin (a macrolide antibiotic) may be at least partially beneficial [121]. In some studies, treatment with nitazoxanide significantly shortened the duration of diarrhea and decreased the risk of mortality in malnourished children [122], and clinical trials showed efficacy in adults [123]. However, other studies showed that nitazoxanide therapy failed to exhibit activity in immunocompromised patients [124,125]. Since the studies showing effectiveness in patients come from one sole source, it is questionable whether there is at all efficacy of nitazoxanide against cryptosporidiosis in patients. More promising, elevating CD4+ T-cell levels in HIV-infected patients by highly active antiretroviral therapy has led to the cessation of life-threatening cryptosporidial diarrhea [79]. This improvement is likely to result from immune reconstitution but may, in part, also be due to the activity of protease inhibitors. Thus, for therapy in HIV patients, nitazoxanide or paromomycin combined with azithromycin should be considered along with initiating antiretroviral therapy [79,83]. Currently, more promising compounds are being developed [126], including inhibitors of inosine monophosphate dehydrogenase [127], calcium-dependent protein kinase I [128], and lipid kinase [129] as the most striking examples. Pathogens 2019, 8, 116 13 of 21

4.4. Is Vaccination a Suitable Strategy? Over the last two centuries, vaccination has proven most useful in fighting against a plethora of life-threatening infective agents. Therefore, it is reasonable that many groups invest in developing vaccination tools and strategies against intestinal protozoan pathogens. In the case of giardiasis, these attempts are hampered by “antigenic switch” (see above). To circumvent this problem, a potential vaccine strain expressing multiple variant surface proteins (VSPs) on a single cell has been created by disrupting the RNA interference mechanism silencing VSP expression [130]. Allegedly, experimentally infected gerbils can be protected against subsequent infections by “vaccination” with this strain or by immunization with recombinant VSPs. Cryptosporidiosis can be prevented by vaccination with lyophilized oocysts, as shown for calves [131]. This result is of particular interest for the poultry industry where the closely related Eimeria constitute a major problem [132]. As a consequence, there is a constant and continuing effort to identify suitable candidates such as, for example, protein p23 [133], a rhomboid protein [134], or surface glycoproteins [135] as vaccine candidates, either as recombinant proteins, DNA vaccine or expressed in a suitable bacterial strain [136]. Vaccination strategies against both diseases may work in suitable animal models and may even have some relevance for farm animals, but in the case of human patients, they are hampered by the fact that the subpopulations with the highest exposure and the highest risk such as malnourished children and HIV-positive adults are immunocompromised. As consequence, the immune responses elicited by recombinant vaccines are impaired [98]. A successful vaccine candidate against giardiasis should comprise the whole pattern of VSPs that may be expressed on an infective strain. This is impossible since the genomes of the sequenced Giardia strains contain several hundred VSP genes, and not all pathogenic strains are amenable to culture and, thus, to sequencing and characterization of their VSP patterns. Clearly, vaccination seems to be a more promising strategy against amebiasis [137]. Vaccination with the Gal-lectin [138] successfully prevents amebiasis in gerbils [139,140], baboons [141], and as a subunit vaccine in a mouse model [142]. Moreover, other suitable vaccine candidates have been identified in the Entamoeba genome [143]. The challenge is to pass from a suitable animal model to tests in humans.

5. Conclusions and Outlook Taken together, intestinal protozoan pathogens, in particular G. lamblia, Cryptosporidium sp., and E. histolytica, cause disabling and even life-threatening diseases. Transmitted by cysts, they can be controlled through water treatment. Antigiardial chemotherapy is well established and alternatives to the main lines of treatment are available. Against cryptosporidiosis, suitable treatment options are, however, lacking and constitute an interesting field for future investigations. Amebiasis, the most lethal of the three, is also the most difficult to treat. Here, the development of both chemotherapy and vaccination will be one of the most challenging issues in parasitology for the future. Another interesting field for investigations would be the influence of overcome giardiasis, cryptosporidiosis, and infection by non-invading ameba on the gastrointestinal absorption of nutrients, the microflora, and the immune system [31].

Author Contributions: All three authors equally contributed to the conceptualization and the writing of the present article. Funding: This work was funded by the Swiss National Science Foundation grants 31003A_163230 and 31003A_184662. Acknowledgments: We thank Larissa Hofmann (Institute of Parasitology, Bern) for providing micrographs of modified Ziehl–Neelsen stained oocyst preparations, Philipp Olias (Institute of Animal Pathology, University of Bern) for providing histological sections of C. parvum-infected intestinal tissue, and Dominic Ritler (Institute of Parasitology, Bern) for designing parts of the illustrations. Conflicts of Interest: The authors declare no conflict of interest. Pathogens 2019, 8, 116 14 of 21

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